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Transcription Precedes Loss of Xist Coating and Depletion of H3k27me3 During X-Chromosome Reprogramming in the Mouse Inner Cell Mass Lucy H

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Transcription Precedes Loss of Xist Coating and Depletion of H3k27me3 During X-Chromosome Reprogramming in the Mouse Inner Cell Mass Lucy H RESEARCH ARTICLE 2049 Development 138, 2049-2057 (2011) doi:10.1242/dev.061176 © 2011. Published by The Company of Biologists Ltd Transcription precedes loss of Xist coating and depletion of H3K27me3 during X-chromosome reprogramming in the mouse inner cell mass Lucy H. Williams, Sundeep Kalantry*, Joshua Starmer and Terry Magnuson† SUMMARY Repression of Xist RNA expression is considered a prerequisite to reversal of X-chromosome inactivation (XCI) in the mouse inner cell mass (ICM), and reactivation of X-linked genes is thought to follow loss of Xist RNA coating and heterochromatic markers of inactivation, such as methylation of histone H3. We analyzed X-chromosome activity in developing ICMs and show that reactivation of gene expression from the inactive-X initiates in the presence of Xist coating and H3K27me3. Furthermore, depletion of Xist RNA coating through forced upregulation of NANOG does not result in altered reactivation kinetics. Taken together, our observations suggest that in the ICM, X-linked gene transcription and Xist coating are uncoupled. These data fundamentally alter our perception of the reactivation process and support the existence of a mechanism to reactivate Xp-linked genes in the ICM that operates independently of loss of Xist RNA and H3K27me3 from the imprinted inactive-X. KEY WORDS: X inactivation, Epigenetic reprogramming, Pre-implantation mouse development INTRODUCTION gradually coats the Xp during pre-implantation development X-chromosome inactivation (XCI) results in the transcriptional (Huynh and Lee, 2003; Okamoto et al., 2004). By the early silencing of most genes on one of the two X-chromosomes to blastocyst stage, prior to specification of extra-embryonic and equalize X-linked gene dosage in female cells with that in XY male embryonic lineages, Xist accumulation on the Xp (Xp-Xist coating) cells (Avner and Heard, 2001). Although the exact mechanism for is evident in all cells of the embryo (Sheardown et al., 1997). At X-linked gene silencing is unknown, XCI requires expression of the late blastocyst stage, when extra-embryonic and embryonic the long non-coding RNA X inactive-specific transcript (Xist) lineages are determined, Xp-Xist coating is maintained in the extra- (Brown et al., 1991; Marahrens et al., 1997; Penny et al., 1996). embryonic trophectoderm and primitive endoderm. By contrast, Xist RNA is expressed from and preferentially coats the inactive X- Xp-Xist coating is lost in epiblast cells, which define the progenitor chromosome (Clemson et al., 1996). Xist coating is followed by cells of the embryo proper (Mak et al., 2004; Okamoto et al., 2004; recruitment of multiple chromatin-modifying complexes, such as Sheardown et al., 1997). the polycomb repressive complex 2 (PRC2), which trimethylates Based on patterns of Xp-Xist coating in the blastocyst, it is lysine 27 on histone H3 to give H3K27me3 (Plath et al., 2002; assumed that all cells of the pre-implantation embryo first undergo Silva et al., 2003). H3K27me3 is a chromatin modification the imprinted form of XCI and that this imprint is maintained in the associated with gene repression, and H3K27me3 enrichment on the extra-embryonic lineages. In the developing ICM, epiblast inactive-X is essential to maintain imprinted paternal-X silencing precursor cells reverse imprinted XCI in order to subsequently in extra-embryonic lineages (Kalantry et al., 2006; Simon and initiate random XCI later in development (Mak et al., 2004; Kingston, 2009). Together, Xist coating and H3K27me3 are Okamoto et al., 2004). However, three recent reports analyzed Xp- necessary to establish stable and heritable XCI; thus, these linked gene transcription in single cells of pre-implantation epigenetic modifications are considered to be markers of X-linked embryos. Each report independently confirmed that some degree gene silencing. of Xp-linked gene silencing is evident as early as the four-cell Two types of XCI are evident during development of the female cleavage stage. Yet, despite Xist coating the Xp in all cells, Xp- mouse embryo. The first type, imprinted XCI, results in exclusive linked gene silencing is not evident in all cells of the blastocyst, silencing of the paternal X-chromosome (Takagi and Sasaki, 1975; indicating that imprinted XCI is incomplete during pre- West et al., 1977). By contrast, the second type of XCI is termed implantation development (Kalantry et al., 2009; Namekawa et al., random because either maternal-X (Xm) or paternal-X (Xp) can 2010; Patrat et al., 2009). Given these results, it is still unclear be chosen for silencing (Lyon, 1961). During early mouse whether epiblast progenitor cells, which are specified by the embryogenesis, Xist expression is first evident at the two-cell stage blastocyst stage, exhibit imprinted Xp-linked gene silencing. and occurs exclusively from the Xp (Kay et al., 1993). Xist RNA Here, we utilized RNA fluorescence in situ hybridization (FISH) techniques to measure directly the kinetics of X-linked gene Department of Genetics, Carolina Center for Genome Sciences, and Lineberger expression, as opposed to chromosome-wide markers of Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599- inactivation, in individual nuclei of developing ICMs. We provide 7264, USA. evidence that supports previous assumptions that, prior to epiblast *Present address: Department of Human Genetics, 1241 E. Catherine St, University specification, ICM cells exhibit imprinted XCI. We also make the of Michigan Medical School, Ann Arbor, MI 48109-5618, USA surprising observation that reactivation in the ICM occurs earlier †Author for correspondence ([email protected]) than previously indicated by loss of Xist coating (Mak et al., 2004; Accepted 17 February 2011 Okamoto et al., 2004). Coincident with specification of the Nanog- DEVELOPMENT 2050 RESEARCH ARTICLE Development 138 (10) expressing epiblast lineage, but prior to loss of the Xist domain and extra-embryonic endoderm (XEN) cell lines, respectively. For combined H3K27me3 enrichment on the Xp, mid-stage ICMs exhibit an immunostaining of H3K27me3 and RNA FISH, RNA FISH was performed increase in Xp-linked gene expression, indicative of active after immunostaining. transcription. Recent results suggest that pluripotency-associated Statistical analysis of RNA FISH transcription factors NANOG, OCT3/4 (POU5F1 – Mouse RNA FISH nuclei were classified as biallelic, monoallelic or nonscorable. Genome Informatics) and SOX2 might directly regulate X- Nuclei were classified as nonscorable when no signal could be detected. chromosome activity by preventing upregulation of Xist expression For each X-linked gene, the total number of the three classes of nuclei was in mouse embryonic stem cells (ES cells) (Navarro et al., 2008). compared between early and mid-stage ICMs. Binomial exact test was used We show that forced upregulation of NANOG results in loss of Xp- to verify that nonscorable data between stages were equivalent. Fisher’s Xist coating without affecting the kinetics of Xp-linked gene exact test compared the three categories between early and mid-stage reactivation. These observations argue against a direct role for ICMs. P values <0.01 were considered to be statistically significant. The procedure was repeated for the Cot-1 RNA FISH experiments except nuclei NANOG-dependent Xist repression as a requirement for initiation were scored as either excluded, overlapping or nonscorable. of Xp expression. We present an alternative hypothesis that reversal of imprinted X-chromosome inactivation occurs by separable steps, Microscopy with gene reactivation being independent from loss of the Xist and Images for RNA FISH were obtained using a Leica DML fluorescence H3K27me3 domains. microscope with a Q-imaging Retiga 200R camera and Q-capture software. Images were processed using SPOT RT Software (Diagnostic Instruments). MATERIALS AND METHODS For Cot-1 RNA FISH and immunostaining, a Zeiss710 confocal microscope was used. Confocal sections were analyzed using Zen software Embryo collection and immunosurgery to determine overlap between signals. To determine relative intensity Embryos were collected from natural mating of either Mus musculus values obtained in the Cot-1 RNA experiments, we generated line traces domesticus CD1 females (RNA FISH) or Mus musculus molossinus JF1 across nuclei using NIH Image J software and used the plot profile tool to females (RT-PCR) to CD1 males carrying the X-GFP (D4.EGFP) transgene measure the intensity across the line traces. (Hadjantonakis et al., 1998). Female blastocysts were distinguished from –/– males by GFP expression. The Grb2 (Grb2tm2Paw) line was described RT-PCR previously (Cheng et al., 1998). Mice were exposed to light daily between RT-PCR was performed as described previously (Kalantry et al., 2009). In 06.00 h and 18.00 h. The morning of the day the plug was detected was brief, mRNA was prepped using the Dynabeads mRNA DIRECT Micro considered to be embryonic day (E)0.5. Early blastocysts were collected Kit (Invitrogen, 610.21). ICMs were lysed in 100 l of lysis/binding buffer. between E2.75 and E3.0 (~66-72 hours post-copulation). Mid-stage For RT-PCR, SuperScript III One-Step RT-PCR Platinum Taq (Invitrogen, blastocysts were collected between E3.5 and E3.75 (~84-90 hours post- 12574-035) was used to amplify the cDNA. A portion of the final RT-PCR copulation). Because embryos collected at the same time varied in product was used to perform one round of amplification in the presence of developmental stage,
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